Three types of human glutathione peroxidase have been characterized: plasma glutathione peroxidase (pl.GPx), intracellular glutathione peroxidase (c.GPx) which is essentially active on hydrophilic substrates, and glutathione peroxidase which is active on phospholipid hydroperoxides as well as on other lipid substrates (PHGPx). These enzymes catalyse the reduction of hydroperoxides (H.sub.2 O.sub.2 or ROOH) by glutathione (GSH). These enzymes are selenoproteins which contain a selenocysteine within their active site. c.GPx and pl.GPx are homotetramers whereas PHGPx is exclusively monomeric Biochem. Biophys. Acta, 839, 62-70 (1985).
Originally purified from plasma J. Biol. Chem., 262, No.36, 17398-17403 (1987); Arch. Biochem. Biophys., 256,No.2, 677-686 (1987), pl.GPx represents 0.007% of the total plasma protein mass. pl.GPx has also been isolated from human maternal milk J. Nutr., 121, No.8, 1243-1249 (1991).
Of glycoprotein nature, pl.GPx exists in the form of a homotetramer of a molecular weight of 94 kDa J. Biol. Chem., 262, No.36, 17398-17403 (1987). Each subunit, characterized by a molecular weight of 21.5 to 23 kDa J. Biol. Chem., 262, No.36, 17398-17403 (1987); J. Biochem., 108, 145-148 (1990), contains a selenocysteine in its active site.
Recently, a study performed in rats has shown that pl.GPx is predominantly synthesized by the renal cell (Yoshimura et al. J. Biochem. 109; (1991); 918-923) whereas another study has shown that a tumorous hepatic cell line (Hep G2) of human origin is capable of synthesizing this pl.GPx (AVISSAR et al. J. Biol. Chem. 264; 1989; 15850-15855).
Given the low concentrations of plasma glutathione, the principal role of pl.GPx remains uncertain. The specific activity of pl.GPx is about ten times lower than that of c.GPx Arch. Biochem. Biophys., 256, No.2, 677-686 (1987).
The primary sequence of human pl.GPx was determined from the cDNA and published by Takahashi et al. J. Biochem., 108; (1990); 145-148. It was not possible to completely sequence the pl.GPx and to identify the N-terminal end because of the glycosylation affecting this end. The exact number of residues per subunit therefore remains unknown.
pl.GPx differs considerably from c.GPx from the structural point of view:. absence of glycosylation and intramolecular disulphide bridges for the cellular form Arch. Biochem. Biophys., 256, 677-686 (1987); Blood, 73, 318-323 (1989), weak sequence homology between the two enzymes (of the order of 44% Nucleic Acids Res., 15; (1987); 5484; Nucleic Acids Res., 15; (1987); 7178).
Measurement of the pl.GPx level in plasma is a good indicator of a possible state of selenium deficiency in the body. Within the framework of experiments designed to quantify the influence of a deficiency and then of a supply of selenium in man, it has been shown that the average glutathione peroxidase levels do not vary at the same rate depending on whether the plasma form or the cellular form is involved. Thus, the pl.GPx level in plasma begins to increase from the early days of treatment to reach a normal value after two to four weeks. The time necessary for the value of the normal level of c.GPx to be reestablished is longer: three to four months Amer. J. Clin. Nutr., 41; (1985); 735-747.
Furthermore, and following the studies previously carried out in rats, a number of renal pathologies could be detected by the measurement of pl.GPx.
None of the known methods of assaying enzymatic activity currently permit the specific assay of pl.GPx in biological fluids or in tissue extracts.
The most commonly used method of assaying glutathione peroxidase activity involves an enzymatic coupling calling into play the regeneration of reduced glutathione by the glutathione reductase present in excess; this reaction is accompanied by the oxidation of a reduced cofactor, NADPH, whose rate of disappearance can be easily monitored as a function of time. Under these conditions, the kinetics of oxidation of NADPH corresponds to the kinetics of oxidation of glutathione and, consequently, to the kinetics of reduction of hydroperoxide. However, no hydroperoxide substrate specific for pl.GPx or c.GPx exists.
Discrimination between the activity of c.GPx and the activity of pl.GPx is therefore impossible at the present time.
Given the fact that the specific activity of c.GPx is approximately ten times greater than that of pl.GPx, a contamination of 1% by mass by c.GPx will introduce an overestimation of the order of 10% of the pl.GPx activity. A slight haemolysis occurring in a blood sample can thus introduce a large error into the calculation of the pl.GPx activity of the circulating plasma or the serum.